pmt3 p38 (Addgene inc)

Addgene inc pmt3 p38

FIG. 5. PEST domain controls the phosphorylation of <t>p38</t> MAPK. Serum-starved PAE cells expressing VEGFR-2, PEST()VEGFR-2, and PEST()VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and blotted for phospho-p38 (A), total p38 (B), phospho-PLC1 (D), and total PLC1 (E). (C) Quantiﬁcation of activation of p38. Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and immunoblotted for phospho-p38 (F), total p38 (G), phospho-PLC1 (H), and total PLC1 (I). Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were preincubated with cycloheximide for 90 min, and then cells were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were immunoblotted for VEGFR-2 (J) and total PLC1 (K). (L) Quantiﬁcation of VEGFR-2 protein levels from blot I is shown. The graph shows averages from two independent experiments. HEK293 cells coexpressing F1173/VEGFR-2 with an empty vector or constitutive active MKK6 (MKK6-Glu) were stimulated with VEGF for the indicated periods of time, and whole-cell lysates were immunoblotted for VEGFR-2 (M), phospho-p38 MAPK (pT180/pY182) (N), p38 MAPK (O), and MKK6 using anti-Flag antibody (P).

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FIG. 5. PEST domain controls the phosphorylation of p38 MAPK. Serum-starved PAE cells expressing VEGFR-2, PEST()VEGFR-2, and PEST()VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and blotted for phospho-p38 (A), total p38 (B), phospho-PLC1 (D), and total PLC1 (E). (C) Quantiﬁcation of activation of p38. Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and immunoblotted for phospho-p38 (F), total p38 (G), phospho-PLC1 (H), and total PLC1 (I). Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were preincubated with cycloheximide for 90 min, and then cells were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were immunoblotted for VEGFR-2 (J) and total PLC1 (K). (L) Quantiﬁcation of VEGFR-2 protein levels from blot I is shown. The graph shows averages from two independent experiments. HEK293 cells coexpressing F1173/VEGFR-2 with an empty vector or constitutive active MKK6 (MKK6-Glu) were stimulated with VEGF for the indicated periods of time, and whole-cell lysates were immunoblotted for VEGFR-2 (M), phospho-p38 MAPK (pT180/pY182) (N), p38 MAPK (O), and MKK6 using anti-Flag antibody (P).

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 5. PEST domain controls the phosphorylation of p38 MAPK. Serum-starved PAE cells expressing VEGFR-2, PEST()VEGFR-2, and PEST()VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and blotted for phospho-p38 (A), total p38 (B), phospho-PLC1 (D), and total PLC1 (E). (C) Quantiﬁcation of activation of p38. Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time, cells were lysed, and whole-cell lysates were subjected to Western blot analysis and immunoblotted for phospho-p38 (F), total p38 (G), phospho-PLC1 (H), and total PLC1 (I). Serum-starved PAE cells expressing VEGFR-2 and F1173/VEGFR-2 were preincubated with cycloheximide for 90 min, and then cells were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were immunoblotted for VEGFR-2 (J) and total PLC1 (K). (L) Quantiﬁcation of VEGFR-2 protein levels from blot I is shown. The graph shows averages from two independent experiments. HEK293 cells coexpressing F1173/VEGFR-2 with an empty vector or constitutive active MKK6 (MKK6-Glu) were stimulated with VEGF for the indicated periods of time, and whole-cell lysates were immunoblotted for VEGFR-2 (M), phospho-p38 MAPK (pT180/pY182) (N), p38 MAPK (O), and MKK6 using anti-Flag antibody (P).

Article Snippet: The following plasmids were purchased from Addgene: pcDNA3-EGFP-Cdc42(WT) and pcDNA3-EGFP-Cdc42 (3); pCDNA3-Flag MKK6(glu), pCDNA3-Flag MKK6, and pCDNA3-Flag MKK6(K82A) (30); pCalpha EV (PKA catalytic subunit C ) (44); M7pdnPKA-GFP (45); and pCDNA3 Flag p38 (agf) pCDNA3 Flag p38 2 and pCDNA3 Flag p38 2 (7). pMT3 p38 (from the J. Kyriakis laboratory) and hemagglutinin (HA) GSK3 S9A pcDNA3 (40) also were used.

Techniques: Phospho-proteomics, Expressing, Western Blot, Activation Assay, Plasmid Preparation

FIG. 6. MKK6-dependent activation of p38 inhibits downregulation of VEGFR-2. HEK 293 cells coexpressing VEGFR-2 with an empty vector, VEGFR-2 with wild-type p38 MAPK, or VEGFR-2 with dominant-negative p38 (dn-P38) MAPK were preincubated with cycloheximide for 90 min and then stimulated with VEGF for the indicated periods of time. Whole-cell lysates were blotted for VEGFR-2 (A), PLC1 as a control for protein loading (B), and p38 (C). (D) The quantiﬁcation of the downregulation of VEGFR-2 in response to ligand stimulation. Whole-cell lysates from HEK293 cells coexpressing VEGFR-2 either with an empty vector, constitutive active MKK6 (MKK6-Glu), or with dominant-negative MKK6 (MKK6-Ala) were immunoblotted for VEGFR-2 (E), phospho-VEGFR-2 (pY1054-VEGFR-2) (F), phospho-p38 MAPK (pT180/pY182) (G), p38 MAPK (H), and MKK6 using anti-Flag antibody (I). HEK293 cells expressing VEGFR-2 were transfected either with empty vector or with enhanced green ﬂuorescent protein (EGFP)-tagged Cdc42. Serum-starved cells were stimulated with VEGF for the indicated times, and cells were lysed. Whole-cell lysates were blotted for VEGFR-2 (J), phospo-p38 (K), total p38 (L), and anti-GFP for Cdc42 expression (M). Cells also were treated with cycloheximide (20 mM for 90 min prior to stimulation with VEGF. HUVEC were transfected with control (Ctr.) siRNA or p38 siRNA after 24 h, and cells were starved overnight and stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were blotted with anti-VEGFR-2 antibody (N), anti-Hsp70 antibody (O), and anti-p38 antibody (P). (Q) Ubiquitination of VEGFR-2 in HUVEC in which p38 was silenced. (S) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates were blotted for p38 (R) and Hsp90 (T) for protein loading.

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 6. MKK6-dependent activation of p38 inhibits downregulation of VEGFR-2. HEK 293 cells coexpressing VEGFR-2 with an empty vector, VEGFR-2 with wild-type p38 MAPK, or VEGFR-2 with dominant-negative p38 (dn-P38) MAPK were preincubated with cycloheximide for 90 min and then stimulated with VEGF for the indicated periods of time. Whole-cell lysates were blotted for VEGFR-2 (A), PLC1 as a control for protein loading (B), and p38 (C). (D) The quantiﬁcation of the downregulation of VEGFR-2 in response to ligand stimulation. Whole-cell lysates from HEK293 cells coexpressing VEGFR-2 either with an empty vector, constitutive active MKK6 (MKK6-Glu), or with dominant-negative MKK6 (MKK6-Ala) were immunoblotted for VEGFR-2 (E), phospho-VEGFR-2 (pY1054-VEGFR-2) (F), phospho-p38 MAPK (pT180/pY182) (G), p38 MAPK (H), and MKK6 using anti-Flag antibody (I). HEK293 cells expressing VEGFR-2 were transfected either with empty vector or with enhanced green ﬂuorescent protein (EGFP)-tagged Cdc42. Serum-starved cells were stimulated with VEGF for the indicated times, and cells were lysed. Whole-cell lysates were blotted for VEGFR-2 (J), phospo-p38 (K), total p38 (L), and anti-GFP for Cdc42 expression (M). Cells also were treated with cycloheximide (20 mM for 90 min prior to stimulation with VEGF. HUVEC were transfected with control (Ctr.) siRNA or p38 siRNA after 24 h, and cells were starved overnight and stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates were blotted with anti-VEGFR-2 antibody (N), anti-Hsp70 antibody (O), and anti-p38 antibody (P). (Q) Ubiquitination of VEGFR-2 in HUVEC in which p38 was silenced. (S) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates were blotted for p38 (R) and Hsp90 (T) for protein loading.

Techniques: Activation Assay, Plasmid Preparation, Dominant Negative Mutation, Control, Expressing, Transfection, Ubiquitin Proteomics, Membrane

FIG. 7. p38 is not involved in the ubiquitination of VEGFR-2. HEK293 cells expressing VEGFR-2 were transfected with empty vector, GSK3, dominant-negative p38 (Dn/p38), wild-type p38, dominant-negative PKA catalytic subunit C (Dn/PKA), and wild-type PKA catalytic subunit C [PKA(C)]. Cells were stimulated with VEGF for 10 min, lysed, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with antiubiquitin (Ub; FK2) antibody (A). (B) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates (WCL) from panel A were blotted with anti-phospho-Ser1188 antibody (C), anti-VEGFR-2 antibody (D), anti-GSK3 antibody (E), anti-p38 antibody (F), anti-GFP antibody to detect dominant-negative PKA (G), anti-PKA antibody (H), and anti-PLC1 antibody (I). HEK293 cells expressing VEGFR-2 were transfected with GST-tagged -Trcp1 alone or GST-tagged -Trcp1 with GSK3, -Trcp1 with p38, or -Trcp1 with p38 and GSK3. (J) Cells were stimulated with VEGF for 10 min, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with anti-ubiquitin (FK2) antibody. (K) The same membrane was reblotted for VEGFR-2. (L to N) Whole-cell lysates from panel J were blotted for GSK3, p38, and -Trcp1.

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 7. p38 is not involved in the ubiquitination of VEGFR-2. HEK293 cells expressing VEGFR-2 were transfected with empty vector, GSK3, dominant-negative p38 (Dn/p38), wild-type p38, dominant-negative PKA catalytic subunit C (Dn/PKA), and wild-type PKA catalytic subunit C [PKA(C)]. Cells were stimulated with VEGF for 10 min, lysed, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with antiubiquitin (Ub; FK2) antibody (A). (B) The same membrane was reblotted for VEGFR-2 levels. Whole-cell lysates (WCL) from panel A were blotted with anti-phospho-Ser1188 antibody (C), anti-VEGFR-2 antibody (D), anti-GSK3 antibody (E), anti-p38 antibody (F), anti-GFP antibody to detect dominant-negative PKA (G), anti-PKA antibody (H), and anti-PLC1 antibody (I). HEK293 cells expressing VEGFR-2 were transfected with GST-tagged -Trcp1 alone or GST-tagged -Trcp1 with GSK3, -Trcp1 with p38, or -Trcp1 with p38 and GSK3. (J) Cells were stimulated with VEGF for 10 min, immunoprecipitated with anti-VEGFR-2 antibody, and blotted with anti-ubiquitin (FK2) antibody. (K) The same membrane was reblotted for VEGFR-2. (L to N) Whole-cell lysates from panel J were blotted for GSK3, p38, and -Trcp1.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Plasmid Preparation, Dominant Negative Mutation, Immunoprecipitation, Membrane

FIG. 8. PKA pathway inhibits downregulation of VEGFR-2 and activates p38. PAE cells expressing wild-type VEGFR-2 were pretreated with forskolin (40 M) and then stimulated with VEGF for the indicated time periods. Cells were treated with cycloheximide for 90 min to inhibit protein synthesis. Whole-cell lysates were blotted with anti-VEGFR-2 antibody (A) and with anti-PLC1 antibody for protein loading (B). (C) The quantiﬁcation of VEGFR-2 downregulation in the presence or absence of forskolin is shown. Serum-starved HEK293 cells coexpressing VEGFR-2 either an empty vector or the catalytic active subunit (C) of PKA were pretreated with cycloheximide (CHX) for 90 min, and then cells were stimulated with VEGF for the indicated time periods. Whole-cell lysates were immunoblotted with anti-VEGFR-2 antibody (D), anti-PLC1 antibody (F), and anti-PKA antibody (G). (E) The quantiﬁcation of the downregulation of VEGFR-2. The same cell lysates also were blotted for phospho-p38 (H), total p38 (I), phospho-PLC1 (J), total PLC1 (K), phospho-MAPK42/44 (L), and total PKA (M).

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 8. PKA pathway inhibits downregulation of VEGFR-2 and activates p38. PAE cells expressing wild-type VEGFR-2 were pretreated with forskolin (40 M) and then stimulated with VEGF for the indicated time periods. Cells were treated with cycloheximide for 90 min to inhibit protein synthesis. Whole-cell lysates were blotted with anti-VEGFR-2 antibody (A) and with anti-PLC1 antibody for protein loading (B). (C) The quantiﬁcation of VEGFR-2 downregulation in the presence or absence of forskolin is shown. Serum-starved HEK293 cells coexpressing VEGFR-2 either an empty vector or the catalytic active subunit (C) of PKA were pretreated with cycloheximide (CHX) for 90 min, and then cells were stimulated with VEGF for the indicated time periods. Whole-cell lysates were immunoblotted with anti-VEGFR-2 antibody (D), anti-PLC1 antibody (F), and anti-PKA antibody (G). (E) The quantiﬁcation of the downregulation of VEGFR-2. The same cell lysates also were blotted for phospho-p38 (H), total p38 (I), phospho-PLC1 (J), total PLC1 (K), phospho-MAPK42/44 (L), and total PKA (M).

Techniques: Expressing, Plasmid Preparation

FIG. 9. Tyrosine 1173 of VEGFR-2 is required for PKA phosphorylation but not for its association with VEGFR-2. PAE cells expressing VEGFR-2 or F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates (WCL) were blotted for phospho-PKA (A) and total PKA (B). (C) Quantiﬁcation of the phosphorylation of PKA is shown. It represents averages from two experiments. HEK293 cells were transfected with GFP-tagged PKA catalytic subunit C alone or with Myc-tagged AKAP1. (D) Cells were lysed, immunoprecipitated (Ipt) with anti-GFP antibody, and blotted with anti-Myc antibody. (E) Whole-cell lysates also were blotted for anti-GFP. Serum-starved HEK293 cells coexpressing VEGFR-2 with empty vector or with c-myc-tagged AKAP1 were stimulated with VEGF for the indicated periods of time (F). Cells were lysed, and VEGFR-2 was immunoprecipitated with anti-VEGFR-2 antibody and immunoblotted with anti-c-myc antibody. (G) The same membrane was stripped and reblotted for VEGFR-2. Whole-cell lysates from the same cell groups were immunoblotted for VEGFR-2 (H), total PLC1 (I), phospho-p38 (J), and total p38 (K).

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 9. Tyrosine 1173 of VEGFR-2 is required for PKA phosphorylation but not for its association with VEGFR-2. PAE cells expressing VEGFR-2 or F1173/VEGFR-2 were stimulated with VEGF for the indicated periods of time. Cells were lysed, and whole-cell lysates (WCL) were blotted for phospho-PKA (A) and total PKA (B). (C) Quantiﬁcation of the phosphorylation of PKA is shown. It represents averages from two experiments. HEK293 cells were transfected with GFP-tagged PKA catalytic subunit C alone or with Myc-tagged AKAP1. (D) Cells were lysed, immunoprecipitated (Ipt) with anti-GFP antibody, and blotted with anti-Myc antibody. (E) Whole-cell lysates also were blotted for anti-GFP. Serum-starved HEK293 cells coexpressing VEGFR-2 with empty vector or with c-myc-tagged AKAP1 were stimulated with VEGF for the indicated periods of time (F). Cells were lysed, and VEGFR-2 was immunoprecipitated with anti-VEGFR-2 antibody and immunoblotted with anti-c-myc antibody. (G) The same membrane was stripped and reblotted for VEGFR-2. Whole-cell lysates from the same cell groups were immunoblotted for VEGFR-2 (H), total PLC1 (I), phospho-p38 (J), and total p38 (K).

Techniques: Phospho-proteomics, Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, Membrane

FIG. 10. Proposed model for PEST-mediated downregulation of VEGFR-2. Upon ligand binding, the PEST domain of VEGFR-2 is phos- phorylated on tyrosine and serine sites, including Y1173, S1188, and Ser1191. Ligand binding promotes -Trcp1 association with VEGFR-2, which mediates the ubiquitination of VEGFR-2 through the Lys-48-dependent ubiquitin chain, leading to degradation by the 26S proteasome system. The activation of PKA by VEGFR-2 requires the AKAP1-mediated association of PKA with VEGFR-2, and its phosphorylation is mediated through Y1173 of VEGFR-2. The activation of PKA leads to the phosphorylation of p38. Activated p38 attenuates the downregulation of VEGFR-2.

Journal: Molecular and Cellular Biology

Article Title: PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

doi: 10.1128/mcb.01006-10

Figure Lengend Snippet: FIG. 10. Proposed model for PEST-mediated downregulation of VEGFR-2. Upon ligand binding, the PEST domain of VEGFR-2 is phos- phorylated on tyrosine and serine sites, including Y1173, S1188, and Ser1191. Ligand binding promotes -Trcp1 association with VEGFR-2, which mediates the ubiquitination of VEGFR-2 through the Lys-48-dependent ubiquitin chain, leading to degradation by the 26S proteasome system. The activation of PKA by VEGFR-2 requires the AKAP1-mediated association of PKA with VEGFR-2, and its phosphorylation is mediated through Y1173 of VEGFR-2. The activation of PKA leads to the phosphorylation of p38. Activated p38 attenuates the downregulation of VEGFR-2.

Techniques: Ligand Binding Assay, Ubiquitin Proteomics, Activation Assay, Phospho-proteomics

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